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Biomedical subjects

C A O'Callaghan

Publications and source records attributed to C A O'Callaghan.

At least 19 recordsLinked to original sources
PubMed2006

Defining the T cell antigen proteome of wasp venom.

BACKGROUND: While modulation of T cell function is believed to be important in the successful acquisition of clinical tolerance during venom immunotherapy, little is known of the role of wasp venom specific T cell antigens. OBJECTIVE: We sought comprehensively to characterize the T cell proteome for wasp venom to facilitate the future development of T cell-based immunotherapeutic approaches. METHODS: Using peripheral blood mononuclear cells from wasp venom-allergic individuals and IL-4 ELISPOT analysis, we characterized T cell responses to whole venom and gel filtration/ion exchange-fractionated venom. Reactive fractions were purified and identified using highly sensitive electrospray ion-trap mass spectrometry. RESULTS: Wasp venom-allergic individuals have detectable whole wasp venom-specific T cells directly ex vivo, which show rapid IL-4 effector function. T cell responses to gel filtration/ion exchange fractionated venom were dominated by responses to phospholipase A(1), hyaluronidase and antigen 5. CONCLUSION: Although it is likely that there are many T cell antigens within wasp venom, the main responses are to proteins coincident with the known IgE-binding proteins.

Adult↗
PubMed2004

Renal manifestations of systemic autoimmune disease: diagnosis and therapy.

Renal involvement is relatively common in certain systemic autoimmune diseases, but can be clinically silent. Active surveillance is, therefore, essential because the early recognition of renal involvement may influence the extent of renal recovery. Blood pressure control is also essential, regardless of the underlying disease. In systemic lupus erythematosus, therapy usually depends on the renal biopsy findings as not all forms of renal involvement respond in the same way. Typically, for aggressive disease, therapy is with steroids and a cytotoxic agent, usually cyclophosphamide initially and then azathioprine. In systemic vasculitis with renal involvement, a similar approach is adopted, therapy including steroids and cyclophosphamide initially and then steroids and azathioprine. With severe fulminant disease, plasma exchange or pulsed intravenous methylprednisolone is added initially. Scleroderma renal crises are managed by blood pressure control using angiotensin-converting enzyme inhibitors and other agents as required. Dialysis and transplantation can be successful in these conditions.

Humans↗
PubMed2002

Characteristics and outcome of membranous nephropathy in older patients.

Many patients with idiopathic membranous nephropathy are elderly, but little is known about the natural or treated history of these patients. We have studied a cohort of 155 patients with membranous nephropathy who were recruited and followed-up over a 20 year period. We have compared the clinical features and outcome of the older (>60 years) and younger age groups. There was a higher incidence of an identifiable cause for the nephropathy in older patients. At presentation with idiopathic disease, older patients were more often hypertensive and had worse renal impairment than the younger cohort, but had a similar levels of proteinuria, hypoalbuminemia and hematuria. Thrombotic complications and minor rheumatological complaints were more common in the older patients. Prognosis for life and renal survival was worse in the older onset patients. Treatment was well tolerated in selected older patients and was associated with a better outcome in those selected for treatment.

Adrenal Cortex Hormones↗
PubMed2001

Molecular competition for NKG2D: H60 and RAE1 compete unequally for NKG2D with dominance of H60.

NKG2D is a potent activating receptor on natural killer cells, T cells, and macrophages. Mouse NKG2D interacts with two cell surface ligands related to class I MHC molecules: RAE1 and H60. We used soluble versions of NKG2D, RAE1, and H60 to characterize their interactions. RAE1 and H60 each bind NKG2D with nanomolar affinities, indicating tighter binding than most cell surface immune interactions, but NKG2D binds to H60 with approximately 25-fold higher affinity than to RAE1. RAE1 and H60 compete directly for occupancy of NKG2D, and, thus, NKG2D can be occupied by only one ligand at a time. The NKG2D-H60 interaction is more temperature dependent and makes greater use of electrostatic interactions than the NKG2D-RAE1 interaction. The distinct thermodynamic profiles provide insights into the different molecular mechanisms of the binding interactions.

Animals↗
PubMed2000

Functionally inert HIV-specific cytotoxic T lymphocytes do not play a major role in chronically infected adults and children.

The highly sensitive quantitation of virus-specific CD8(+) T cells using major histocompatibility complex-peptide tetramer assays has revealed higher levels of cytotoxic T lymphocytes (CTLs) in acute and chronic virus infections than were recognized previously. However, studies in lymphocytic choriomeningitis virus infection have shown that tetramer assays may include measurement of a substantial number of tetramer-binding cells that are functionally inert. Such phenotypically silent CTLs, which lack cytolytic function and do not produce interferon (IFN)-gamma, have been hypothesized to explain the persistence of virus in the face of a quantitatively large immune response, particularly when CD4 help is impaired. In this study, we examined the role of functionally inert CTLs in chronic HIV infection. Subjects studied included children and adults (n = 42) whose viral loads ranged from <50 to >100,000 RNA copies/ml plasma. Tetramer assays were compared with three functional assays: enzyme-linked immunospot (Elispot), intracellular cytokine staining, and precursor frequency (limiting dilution assay [LDA]) cytotoxicity assays. Strong positive associations were observed between cell numbers derived by the Elispot and the tetramer assay (r = 0.90). An even stronger association between tetramer-derived numbers and intracellular cytokine staining for IFN-gamma was present (r = 0.97). The majority (median 76%) of tetramer-binding cells were consistently detectable via intracellular IFN-gamma cytokine staining. Furthermore, modifications to the LDA, using a low input cell number into each well, enabled LDAs to reach equivalence with the other methods of CTL enumeration. These data together show that functionally inert CTLs do not play a significant role in chronic pediatric or adult HIV infection.

Adult↗
PubMed2000

Recombinant modified vaccinia virus Ankara efficiently restimulates human cytotoxic T lymphocytes in vitro.

The immunogenicity of recombinant modified vaccinia Ankara, a highly attenuated vaccinia virus, expressing influenza nucleoprotein (MVA-NP) and HIV-1 gag (MVA-gag) was investigated. Restimulation of peripheral blood lymphocytes of healthy subjects with MVA-NP led to expansion of CTL with specificity for known NP epitopes. These CTL efficiently lysed NP peptide-pulsed targets and released interferon-gamma (IFN-gamma) on contact with epitope peptide. MVA-NP-stimulated CTL specific for the HLA-B8 epitope, NP380-88, stained with a tetrameric complex of HLA-B8 refolded with the NP380-88 peptide and anti-CD8 antibody on flow cytometry. CTL were also elicited from two HIV-1 seropositive donors by restimulation with MVA-HIV-1 gag and showed specificity for immunodominant gag epitopes. These data indicate that restimulation of human CTL with recombinant MVA is effective and suggest that MVA will elicit CTL responses in humans in vivo.

Cell Line↗
PubMed2000

Classical and nonclassical class I major histocompatibility complex molecules exhibit subtle conformational differences that affect binding to CD8alphaalpha.

The cell surface molecules CD4 and CD8 greatly enhance the sensitivity of T-cell antigen recognition, acting as "co-receptors" by binding to the same major histocompatibility complex (MHC) molecules as the T-cell receptor (TCR). Here we use surface plasmon resonance to study the binding of CD8alphaalpha to class I MHC molecules. CD8alphaalpha bound the classical MHC molecules HLA-A*0201, -A*1101, -B*3501, and -C*0702 with dissociation constants (K(d)) of 90-220 microm, a range of affinities distinctly lower than that of TCR/peptide-MHC interaction. We suggest such affinities apply to most CD8alphaalpha/classical class I MHC interactions and may be optimal for T-cell recognition. In contrast, CD8alphaalpha bound both HLA-A*6801 and B*4801 with a significantly lower affinity (>/=1 mm), consistent with the finding that interactions with these alleles are unable to mediate cell-cell adhesion. Interestingly, CD8alphaalpha bound normally to the nonclassical MHC molecule HLA-G (K(d) approximately 150 microm), but only weakly to the natural killer cell receptor ligand HLA-E (K(d) >/= 1 mm). Site-directed mutagenesis experiments revealed that variation in CD8alphaalpha binding affinity can be explained by amino acid differences within the alpha3 domain. Taken together with crystallographic studies, these results indicate that subtle conformational changes in the solvent exposed alpha3 domain loop (residues 223-229) can account for the differential ability of both classical and nonclassical class I MHC molecules to bind CD8.

Amino Acid Sequence↗
PubMed2000

Early highly active antiretroviral therapy for acute HIV-1 infection preserves immune function of CD8+ and CD4+ T lymphocytes.

Highly active antiretroviral therapy (HAART) has been advocated for the management of primary HIV-1 infection without clear understanding of its immunological effects. Here, we demonstrate that early use of HAART during primary infection preserves HIV-specific CD8(+) T cells physically and functionally while HIV-specific T cell help is sustained. We also show that even transient administration of HAART at seroconversion can preserve HIV-specific immunity. In contrast, delayed initiation of HAART is associated with a progressive loss of HIV-specific CD8(+) T cells and absent HIV-specific T cell help. These results imply that HIV-specific T help is damaged during primary HIV-1 infection. Early drug treatment, which preserves this immunity, also preserves HIV-specific CD8(+) T cells. These results have implications for understanding the early pathogenesis of HIV-1 infection and suggest that acute HIV infection should be treated aggressively and as early as possible.

Amino Acid Sequence↗
PubMed2000

Functional characterization of HLA-F and binding of HLA-F tetramers to ILT2 and ILT4 receptors.

HLA-F is a human non-classical MHC molecule. Recombinant HLA-F heavy chain was refolded with 2-microglobulin to form a stable complex. This complex was used as an immunogen to produce a highly specific, high-affinity monoclonal antibody (FG1) that was used to study directly the cellular biology and tissue distribution of HLA-F. HLA-F has a restricted pattern of tissue expression in tonsil, spleen, and thymus. HLA-F could be immunoprecipitated from B cell lines and from HUT-78, a T cell line. HLA-F binds TAP, but unlike the classical human class I molecules, was undetected at the cell surface. HLA-F tetramers stain peripheral blood monocytes and B cells. HLA-F tetramer binding could be conferred on non-binding cells by transfection with the inhibitory receptors ILT2 and ILT4. Surface plasmon resonance studies demonstrated a direct molecular interaction of HLA-F with ILT2 and ILT4. These results, together with structural predictions based on the sequence of HLA-F, suggest that HLA-F may be a peptide binding molecule and may reach the cell surface under favorable conditions, which may include the presence of specific peptide or peptides. At the cell surface it would be capable of interacting with LIR1 (ILT2) and LIR2 (ILT4) receptors and so altering the activation threshold of immune effector cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Natural killer cell surveillance of intracellular antigen processing pathways mediated by recognition of HLA-E and Qa-1b by CD94/NKG2 receptors.

HLA-E binds specifically to MHC class Ia leader peptides in a TAP (transporter associated with antigen processing)-dependent manner. It interacts with CD94/NKG2A receptors on natural killer cells and this inhibits natural killer cell lysis of the cell displaying HLA-E. The crystal structure of HLA-E demonstrates that the specificity of leader peptide binding is a structurally defined intrinsic property of HLA-E.

Animals↗
PubMed2000

Molecular basis of human natural killer cell recognition of HLA-E (human leucocyte antigen-E) and its relevance to clearance of pathogen-infected and tumour cells.

HLA-E (human leucocyte antigen-E) is a conserved class I major histocompatibility molecule which has only limited polymorphism. It binds to the leader peptide derived from the polymorphic classical major histocompatibility molecules HLA-A, HLA-B and HLA-C. This peptide binding is highly specific and stabilizes the HLA-E protein, allowing it to migrate to the cell surface. A functioning TAP (transporter associated with antigen processing) molecule is required to transport these peptides into the endoplasmic reticulum, where they can interact with HLA-E. HLA-E then migrates to the cell surface, where it interacts with CD94/NKG2A receptors on natural killer cells. This interaction inhibits natural killer cell-mediated lysis of a cell displaying HLA-E. If the leader peptide is not present in the endoplasmic reticulum, HLA-E is unstable and is degraded before it reaches the cell surface. In damaged cells, such as virally infected or tumour cells, down-regulation of HLA-A, HLA-B and HLA-C production or inhibition of TAP prevents stabilization of HLA-E by the leader peptide. Under these circumstances, HLA-E does not reach the cell surface and the cell is then vulnerable to lysis by natural killer cells. The molecular mechanisms underlying this function of HLA-E have been revealed by crystallographic studies of the structure of HLA-E.

Crystallography↗
PubMed2000

Differential narrow focusing of immunodominant human immunodeficiency virus gag-specific cytotoxic T-lymphocyte responses in infected African and caucasoid adults and children.

Cytotoxic T-lymphocyte (CTL) activity plays a central role in control of viral replication and in determining outcome in cases of human immunodeficiency virus type 1 (HIV-1) infection. Incorporation of important CTL epitope sequences into candidate vaccines is, therefore, vital. Most CTL studies have focused upon small numbers of adult Caucasoid subjects infected with clade-B virus, whereas the global epidemic is most severe in sub-Saharan African populations and predominantly involves clade-C infection in both adults and children. In this study, sensitive enzyme-linked immunospot (elispot) assays have been utilized to identify the dominant Gag-specific CTL epitopes targeted by adults and children infected with clade-B or -C virus. Cohorts evaluated included 44 B-clade-infected Caucasoid American and African American adults and children and 37 C-clade-infected African adults and children from Durban, South Africa. The results show that 3 out of 46 peptides spanning p17(Gag) and p24(Gag) sequences tested contain two-thirds of the dominant Gag-specific epitopes, irrespective of the clade, ethnicity, or age group studied. However, there were distinctive differences between the dominant responses made by Caucasoids and Africans. Dominant responses in Caucasoids were more often within p17(Gag) peptide residues 16 to 30 (38 versus 12%; P < 0.01), while p24(Gag) peptide residues 41 to 60 contained the dominant Gag epitope more often in the African subjects tested (39 versus 4%; P < 0.005). Within this 20-mer p24(Gag), an epitope presented by both B42 and B81 is defined which represents the dominant Gag response in >30% of the total infected population in Durban. This epitope is closely homologous with dominant HIV-2 and simian immunodeficiency virus Gag-specific CTL epitopes. The fine focusing of dominant CTL responses to these few regions of high immunogenicity is of significance to vaccine design.

Adult↗
PubMed1999

Cutting edge: HLA-B27 can form a novel beta 2-microglobulin-free heavy chain homodimer structure.

HLA-B27 has a striking association with inflammatory arthritis. We show that free HLA-B27 heavy chains can form a disulfide-bonded homodimer, dependent on residue Cys67 in their extracellular alpha 1 domain. Despite the absence of beta 2-microglobulin, HLA-B27 heavy chain homodimers (termed HC-B27) were stabilized by a known peptide epitope. HC-B27 complexes were recognized by the conformation-specific Ab W6/32, but not the ME1 Ab. Surface labeling and immunoprecipitation demonstrated the presence of similar W6/32-reactive free heavy chains at the surface of HLA-B27-transfected T2 cells. HC-B27 homodimer formation might explain the ability of HLA-B27 to induce spondyloarthropathy in beta 2-microglobulin-deficient mice.

Antibody Specificity↗
PubMed1999

A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers.

EBV is a gammaherpesvirus that can establish both nonproductive (latent) and productive (lytic) infections within the cells of its host. Although T cell responses to EBV latent proteins have been well characterized, little is known about the importance of responses to lytic proteins in long term virus carriers. Here we have compared the frequencies of CD8+ T cells specific for EBV latent and lytic Ags in healthy virus carriers, using three techniques: limiting dilution analysis, enzyme-linked immunospot assay, and FACS staining with tetrameric MHC-peptide complexes. T cells specific for EBV lytic protein epitopes were readily detectable in all donors and were usually more abundant than those specific for latent epitopes. We infer that direct T cell control of viral replicative lesions is maintained in long term carriers of EBV and is an important component of the immune response to this virus. Estimates of CD8+ T cell frequencies varied considerably according to methodology; values obtained from MHC-peptide tetramer staining were, on the average, 4.4-fold higher than those obtained from enzyme-linked immunospot assays, which were, in turn, on the average, 5.3-fold higher than those obtained from limiting dilution analysis. Tetramer staining showed that as many as 5.5% circulating CD8+ T cells in a virus carrier were specific for a single EBV lytic protein epitope. Such values are much greater than previously imagined and illustrate how antigenic challenge from a persistent herpesvirus can influence the composition of the host's CD8+ T cell pool.

Adult↗
PubMed1999

Production of soluble alphabeta T-cell receptor heterodimers suitable for biophysical analysis of ligand binding.

A method to produce alphabeta T-cell receptors (TCRs) in a soluble form suitable for biophysical analysis was devised involving in vitro refolding of a TCR fusion protein. Polypeptides corresponding to the variable and constant domains of each chain of a human and a murine receptor, fused to a coiled coil heterodimerization motif from either c-Jun (alpha) or v-Fos (beta), were overexpressed separately in Escherichia coli. Following recovery from inclusion bodies, the two chains of each receptor were denatured, and then refolded together in the presence of denaturants. For the human receptor, which is specific for the immunodominant influenza A HLA-A2-restricted matrix epitope (M58-66), a heterodimeric protein was purified in milligram yields and found to be homogeneous, monomeric, antibody-reactive, and stable at concentrations lower than 1 microM. Using similar procedures, analogous results were obtained with a murine receptor specific for an influenza nucleoprotein epitope (366-374) restricted by H2-Db. Production of these receptors has facilitated a detailed analysis of viral peptide-Major Histocompatibility Complex (peptide-MHC) engagement by the TCR using both surface plasmon resonance (SPR) and, in the case of the human TCR, isothermal titration calorimetry (ITC) (Willcox et al., 1999). The recombinant methods described should enable a wide range of TCR-peptide-MHC interactions to be studied and may also have implications for the production of other heterodimeric receptor molecules.

Amino Acid Sequence↗
PubMed1998

Oligoclonal expansions of CD8(+) T cells in chronic HIV infection are antigen specific.

Acute HIV infection is associated with a vigorous immune response characterized by the proliferation of selected T cell receptor V beta (BV)-expressing CD8(+) T cells. These 'expansions', which are commonly detected in the peripheral blood, can persist during chronic HIV infection and may result in the dominance of particular clones. Such clonal populations are most consistent with antigen-driven expansions of CD8(+) T cells. However, due to the difficulties in studying antigen-specific T cells in vivo, it has been hard to prove that oligoclonal BV expansions are actually HIV specific. The use of tetrameric major histocompatibility complex-peptide complexes has recently enabled direct visualization of antigen-specific T cells ex vivo but has not provided information on their clonal composition. We have now made use of these tetrameric complexes in conjunction with anti-BV chain-specific monoclonal antibodies and analysis of cytotoxic T lymphocyte lines/clones to show that chronically clonally expanded CD8(+) T cells are HIV specific in vivo.

CD8-Positive T-Lymphocytes↗